Thin coatings applied to the surface of materials can improve the properties of objects dramatically as they allow control of the interaction of a material with its environment. Examples of interface properties which can be controlled by deposition of a thin organic film onto a surface include friction, adhesion, adsorption of molecules from the surrounding environment, or wetting with water or other liquids, control of electroosmotic flow (EOF) in micro-channel and nano-channels.
In the scientific and patent literature there are examples of film formation that occur either by physisorption or by chemisorption. The synergic combination of the two mechanisms was introduced by the patent EP1567569, and by the U.S. application Ser. No. 10/536,306. In these two documents the use of a copolymer made of three distinct components, each with a specific function, was disclosed: i) a monomer that promotes surface adsorption, ii) a monomer that promotes the covalent binding with biomolecules such as DNA and proteins and iii) a third monomer that stabilizes the adsorbed film by covalently reacting with functional groups present on the surface. The use of the copolymer reported in EP1567569, and by the U.S. application Ser. No. 10/536,306 is limited to the production of a coating for the attachment of biological molecules to different surfaces.
The use of poly(dimethylacrylamide)-based copolymers bearing functionalities reactive towards nucleophiles to coat various materials was introduced by the above mentioned patents. The film formed on the surface confers to the support underneath the ability to immobilize bioactive species. The above cited documents disclose the use of a copolymer made of two or more monomers playing different roles. In particular, the polymer backbone (the monomer constituent present in larger amount) promotes polymer physisorption whereas the other functional monomers are chemically reactive with either the material of the surface or biological molecules.
The above mentioned EP patent describes a method to immobilize biological molecules to a glass surface based on the use of copolymers with a long segment of poly(dimethylacrylamide) functionalized either by random incorporation of N-acryloyloxysuccinimide or glycidyl methacrylate comonomers, optionally also incorporating an allyl monomer able to react with surface silanols. Furthermore, Pirri G., et al. (Characterization of a polymeric adsorbed coating for D NA microarray glass slides, Anal Chem., 2004, 76, 1352-8) have suggested that the third monomer could be a silane monomer.
The copoly(dimethylacrylamide-acryolyloxysuccinmide methacryloylpropyltrimethoxysilane)-copoly(DMA-MAPSA-NAS) has been extensively used to attach biological molecules on several materials (Cretich M., et al., High Sensitivity Protein Assays on Microarray Silicon Slides, Anal. Chem., 2009, 81, 5197-5203, Cretich M. et al., Functionalization of poly(dimethylsiloxane) by chemisorption of copolymers: DNA microarrays for pathogen detection, Sens. and Actuat. B-Chem., 2008, 132, 258-264.).
The copolymer previously reported in EP1567569 and in the publications cited above promotes the attachment of biomolecules by exposing functional active esters that are available for reaction with the nucleophiles groups on the protein, peptide or amino-modified DNA. The active ester on the monomer plays, at the same time, the role of stabilizing the coating by a covalent binding with the surface and grafting the bioactive species by reacting with their exposed functionalities. In the above cited paper by Pirri et al. (Anal Chem., 2004, 76, 1352-8) it has been demonstrated that the introduction of a third silane monomer constituent is highly effective in stabilizing the binding of the polymer with the surface.
Polymer for Suppression or Control of Electroosmotic Flow
In most capillary electrophoresis (CE) applications it is necessary to suppress or control electroosmotic flow in order to exploit the potentiality of the technique. In some cases, for instance at high pH, the EOF may be too rapid resulting in the elution of the analytes before separation has occurred. In addition, the negatively charged wall can cause adsorption of cationic solutes through coulombic interactions. An effective modification of surface properties can be achieved by coating the wall with a polymer. Neutral polymers physi- or chemi-sorbed on the capillary wall, strongly decrease EOF by shielding surface charge and by increasing local viscosity. Silica surfaces, exposed to very diluted solutions of certain polymers, develop dense polymer layers which involve hydrogen bonding, monopolar, dipolar or hydrophobic forces. In the past, the author has described polymer coatings with characteristics of irreversibility obtained by adsorption of copolymers of dimethylacrylamide and allylglycidyl ether (U.S. Pat. No. 6,410,668). Polymer-surface, solvent-surface, and polymer-solvent interactions were optimized to increase the stability of the coating. In particular, the molecular weight was found to strongly impact layer stability as the sticking energies per chain increase in proportion to the number of monomer units.
One of the problems of CE, is that the coating is challenged by a number of factors including shear forces, competition by various species present in the running buffer (urea, detergents proteins). Therefore, the strongest and most enduring interfaces possible are preferred.
The publication Cretich M. et al., (Electroosmotic flow suppression in capillary electrophoresis: chemisorption of trimethoxy silane-modified polydimethylacrylamide, Electrophoresis, 2005, 26, 1913) discloses strategies to form irreversible polymer films on a capillary wall that involve polymer physisorption followed by its covalent attachment to the surface.
Two polymers were independently disclosed by the U.S. Pat. No. 6,410,668 and by the Chiari et al., publication (Electrophoresis, 2005, 26, 1913) reported above. In both polymers, dimethylacylamide is present as the surface interacting monomer but they differ from the polymers disclosed in the present invention. The polymer of U.S. Pat. No. 6,410,668 lacks of the silane condensable monomer whereas the polymer of the publication lacks of the chemically active monomer (epoxy group). It was found that the polymer of the U.S. Pat. No. 6,410,668 requires a high molecular weight to form a stable coating whereas the polymer of the publication lacks of robustness, often failing to provide the desired performance.
Adsorpion of Biomoleucles
The adsorption of biomolecules through specific and nonspecific interactions is important in industrial, medical, and diagnostic applications.
An example of the importance of controlling protein adsorption is given by the biosensors that are routinely and increasingly used. In biosensors, specific interactions between biomolecules, such as complementary DNA strands (Joseph Wang, From DNA biosensors to gene chips, Nucleic Acids Research, 2000, Vol. 28, No. 16 3011-3016) or antibody-antigen pairs (B. Leca-Bouvier and L. J. Blum, Biosensors for Protein Detection: A Review, Analytical Letters, Volume 38, Issue 10 2005, pages 1491-1517) are necessary. The accuracy and reliability of these molecular diagnostic devices are currently hampered by their lack of sensitivity due to a low signal-to-noise ratio. A factor that plays a major role in reducing assay sensitivity is the nonspecific adsorption of biomolecules. The tendency of proteins to adsorb non-specifically to most solid surfaces is responsible for this phenomenon. A protein molecule has various hydrophobic or charged domains that can bind strongly with hydrophobic or oppositely charged surfaces. The hydrophobic interaction is particularly prevalent and is the dominant reason for fouling of a surface.
To solve the problems of non specific adsorption of bioactive species, considerable basic and applied researches have been devoted to the surface modification of various materials. A variety of “antifouling” layers have been employed to reduce the amount of nonspecifically adsorbed molecules on surfaces. Poly(ethylene glycol) (PEG), polysaccharides and polyionic layers have all been successfully incorporated as effective antifouling layers. Generally, there are two ways to achieve the surface modification of materials with polymers: physisorption and covalent attachment. Modification through physisorption of a polymer is technologically simpler, in particular, when the film is produce by dip coating. The disadvantage of physisorbed coatings is their stability which is controlled by the hydrodynamic size of the adsorbing chains. In poly-disperse systems, short chains adsorb more rapidly than long ones but long chains are preferred on the surface at equilibrium. This leads to an exchange between long and short chains at intermediate stages of adsorption. In a radical polymerization it may be difficult to control polymer molecular weight and poly-dispersity. This lack of reproducibility on the composition of the polymer chains can translate into a poor reproducibility of some coating characteristics such as long term stability, thickness and swelling behavior. In addition, physisorption is an equilibrium process and desorption can occur under certain circumstances.
In an effort to overcome the problems of coating instability, a new family of polymer-silane, that rapidly adsorb on the wall from ultra-diluted aqueous solutions, was synthesized and used to form a coating on capillary and microfluidic devices. Upon thermal treatment, silyl groups, pending from the backbone, condensate with surface silanols and covalently bind the copolymer to the surface. The results of the investigation on the characteristics of the coating formed by this polymer proves that, by a combination of physisorption and chemisorption, it is possible to dramatically increase coating adhesion strength leading to coatings of longer lifetime
In the present invention, it was surprisingly found that the combination of three specific ingredients in a linear polymer leads to formation of coating with improved performance as the specific polymer composition allows increasing the number of surface anchoring points. For instance, when use in capillary or micro-channel electrophoresis to suppress EOF, poly(dimethylacrylamide) containing silane and glycidyl methacrylate monomers is more strongly attached to the surface making the coating more robust, reproducible and highly performing to suppress EOF. A similar effect could not be obtained by increasing the concentration of the two individual silane and glycidyl methacrylate monomers. In fact, by increasing the silane molar fraction, the polymer becomes too prone to condensation and electrically charged whereas, by increasing the content of epoxy groups, the surface becomes reactive towards biomolecules that are separated in the capillary leading to band broadening and dramatic decrease of the separation performance. Only the synergistic presence of the two monomers, the silane and the epoxy, allows formation of a coating that combines the advantages of low polymer molecular weight and high coating stability. Stable coatings are generated by covalent attachment of polymers to surfaces but they need cumbersome and multistep chemical reactions which may be difficult to control.
The polymer of the invention overcomes problems such as irreproducibility and instability of physisorbed coating. They are particularly advantageous because due to a combination of physic- and chemi-sorption mechanisms, the polymers of the invention can i) suppress/control electroosmotic flow in electrophoresis ii) minimize non specific adsorption of proteins onto coated surfaces and iii) increase hydrophilicity of the surface on which they are applied.